The present invention is directed toward a method and a disposable device for use in an automated solid-phase diagnostic assay. The device is designed to have a plurality of two well pairs, one of each well pairs is where a sample material can be incubated with reagents to perform a solid-phase assay and another in which the results can be read. The reaction mixture is transferred from the first well, the incubation well, to the second well, the read well by a non-contact means using jets of fluid to move the reactants between the two wells. The disposable device has surface features surrounding each well pair that mates with a chemiluminescent reader head in such a way that a light-tight seal is created to allow low-light level measurements. Associated also with each well pair a means for immobilizing and retaining the reaction products, a means for removal of excess reactants and wash solutions and a vent hole to vent air displaced by fluids added into the device.
Techniques for performing an immunoassay are generally known in the art. For example, conventional enzyme immunoassay procedures involve a series of steps wherein an analyte in sample material is initially bound to a corresponding antigen or antibody reagent. A second antigen or antibody is then introduced into the sample which has been labeled with an enzyme or other substance capable of being detected directly or after addition of a suitable reagent such as a chromogenic or fluorogenic substrate or a trigger solution for activating chemiluminescence. The generated signal is then read to indicate the absence or presence of the antigen or the antibody in the sample.
Solid phase immunoassay procedures are preferred over other diagnostic methods because of their specificity and sensitivity as interfering substances can washed away before optical readout.
One form of a conventional solid-phase immunoassay is a "sandwich assay" which involves contacting a test sample suspected of containing an antibody or antigen with a material which has attached to it a protein or another substance capable of binding the antigen or the antibody to the surface of the support. After the antibody or antigen is bound to the support material it is treated with a second antigen or antibody, which is conjugated with an enzyme, a fluorophore or a chemiluminescent label. The second antigen or antibody then becomes bound to the corresponding antibody or antigen on the support. Following one or more washing steps to remove any unbound material in an enzyme immunoassay, an indicator substance, for example, a chromogenic substrate, is added which reads with the enzyme to produce a color change. The color change can be observed visually or more preferably by an instrument to indicate the presence or absence of the antibody or antigen in the sample. For solid-phase fluorescence or chemiluminescence immunoassays, fluorescent labeled moieties can be monitored by using excitation at an appropriate wavelength, while chemiluminescent labeled antigens or antibodies can be followed after reaction by chemically activating the chemiluminescent labels to generate light which can be detected by photometric means.
Many procedures and apparatus have been designed to perform solid-phase immunoassays. U.S. Pat. No. 4,632,910 discloses an apparatus having a porous filter containing a bound receptor for complexing an analyte. In this apparatus an absorbent material is positioned below the porous filter to assist the fluid sample in flowing through the filter. A labeled antibody is then added to the porous filter to detect the presence or absence of the analyte. This approach leads to assays with limited sensitivities as the sample and conjugate incubation takes place on the same matrix. None-specific binding of the sample and conjugate to the porous matrix can occur and contribute to the background of the assay and limits its sensitivity.
In another approach, European Patent Application No. 0131934 discloses an assay device having a plurality of aligned adjacent incubation wells located on its top surface which empty through a filter membrane located above a waste reservoir. U.S. Pat. No. 4,652,533 discloses an assay method using such a device. A solid-phase fluorescent immunoassay reaction mixture is placed in the well and drawn through the membrane by applying reduced pressure to the waste reservoir to separate a solid-phase reaction product from a liquid-phase reactants so that the solid-phase reaction product can be observed. This approach, however, has serious limitations. First, it is limited to use of microparticles as a capture phase. Secondly, the sample, conjugate and microparticles are incubated in the same incubation well that the optical reading takes place. Non-specific binding of sample and conjugate, labeled antigen or antibody, to the membrane filter in the reading well and the wall of the well can occur and contribute to the background of the assay and thus limits its sensitivity. Third, because of using a common vacuum manifold to a plurality of filters, a pinhole in one of the wells will lead to air leaking through this well and no filteration for other wells in the disposable reaction tray. Thirdly, such a disposable device cannot be used for chemiluminescence immunoassay measurements where extreme light tight conditions around each well are required. Other microparticles vacuum filteration devices for immunoassays are available commercially such as Millititer.RTM. Plate from Millipore Corporation, Bedford, Mass.
Other methods for performing a solid-phase immunoassay are disclosed in U.S. Pat. Nos. 4,587,102, and 4,552,839, and European Patent Application 0200381. These references generally disclose procedures for employing particles having a receptor to bind an analyte which is subsequently labeled and deposited on a matrix or other support system. The particles complex is treated with an indicator substance to indicate the presence or absence of an analyte.
While many immunoassay procedures and devices have proved useful, better procedures and devices are continually being sought to improve reliability, efficiency and sensitivity. The present invention provides all of these improvements.